Regulation of NOS3 by Novel Acetylation Sites

Abstract

Nitric oxide (NO) is important for cardiovascular and renal health. NO is primarily produced via the enzymatically catalyzed reaction of L-arginine and oxygen to produce NO and L-citrulline by nitric oxide synthase (NOS) isoforms, NOS1, NOS2, and NOS3. NOS3 was first identified in the endothelium but is also present in the kidney epithelium. Endothelial NOS3 maintains cardiovascular health by preventing platelet aggregation, smooth muscle proliferation, and leukocyte adhesion. Epithelial NOS3 controls water and electrolyte homeostasis by regulating ion transporter activity along the nephron. NOS3 activity is tightly controlled by posttranslational modifications. While phosphorylation sites have been well characterized, less is known about regulation by acetylation. Lysine acetylation is a dynamic posttranslational modification observed in both the nucleus and cytosol to regulate intracellular signaling. Our lab and others have shown NOS3 to be regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites on NOS3 which modulate its activity. Based on posttranslational modification prediction software we chose seven lysines to investigate: K486, K497, K504, K607, K610, K727, and K731. We hypothesized that deacetylation of any or all of these lysine sites increases NOS3-mediated NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation in the media over 48 hours. Constructs containing either K486R, K497R, or K504R did not show a significant increase in NO production compared to WT (WT: 1703±499.3 pmol NO2/mg protein; K486R: 1984 ± 1003 pmol NO2/mg protein; K497: 1528 ± 1032 pmol NO2/mg protein; K504: 4337±576.7 pmol NO2/mg protein; p < 0.05 vs WT). However, mutations of four lysines, K607R, K610R, K727R, and K731R (K607/610/727/731R), dramatically increased NO production (21971 ± 1941 pmol NO2/mg protein; WT: 1703±499.3 pmol NO2/mg protein; p < 0.0001 vs WT). To further identify which of the four lysines are responsible for the observed phenotype, we made two new constructs containing either K607/610R or K727/731R. K607/610R was similar to WT (WT: 2271 ± 729.3 pmol NO2/mg protein; K607/610R: 3523 ± 805.8 pmol NO2/mg protein: p > 0.05), yet mutations of K727/731R demonstrated a large increase in NO production (9431 ± 1772 pmol NO2/mg protein; WT: 2271 ± 729.3 pmol NO2/mg protein; p<0.05). These studies suggest K727 and/or K731 is basally acetylated and deacetylation increases NO production uncovering novel posttranslational modifications which regulate NOS3 activity. Future studies will identify whether K727, K731, or both sites are responsible for the increase in NO production.